In various chemical processes such as fermentation, it is desirable to monitor the progress of specific chemical reactions that take place in a test container. Most of these applications are long-term processes that can last between a few hours to several days. Typical applications include chemical reactions resulting from growth of microorganisms in liquefied samples. Under appropriate environmental conditions (e.g. temperature and adequate growth nutrients), microorganisms grow, metabolize, and chemically change the liquid growth medium that surrounds them. Several chemical indicators, such as dyes and fluorescent reagents may be added to the assay. These indicators are capable of changing their optical characteristics due to the chemical reaction taking place in the test container. For example Bromcresol Purple (Hach Co. Catalog #25432) can serve as a color PH indicator. When fermenting, the growing microorganisms lower the PH of the liquid medium (i.e. becoming more acidic), changing the color of the indicator from purple to yellow. The color variant can be detected with an external optical sensor that dynamically monitors and registers that change as a function of time.
In practical applications it is desirable to monitor actual product samples such as industrial samples (food, beverages, cosmetics, pharmaceuticals, etc.) or medical samples (blood, tissues, urine, etc.). These products can severely mask the optical readings of the indicators. Products such as milk, powders, and blood can interfere or totally alter the optical readings. Even for clear samples such as water, the growing organisms form turbidity in the solution that can also mask the corresponding optical readings.
In order to decrease the product interference, a two chamber test device has been devised, which includes a container having a first or upper chamber defined therein holding a clear test liquid which is open to allow the sample to be tested to be introduced therein, and also having a second or lower chamber defined therein below the upper chamber and also filled with a clear test liquid. The two chambers are separated by a porous barrier such as a membrane, which is configured so that the product as well as the microorganisms cannot penetrate through the porous barrier, while small molecules and ions can diffuse freely between the layers through the porous barrier and be in equilibrium. The color changes can therefore be read in the liquid in the second chamber by an external optical reader since the test liquid in that chamber remains relatively clear.
Both chambers enclose identical mixtures of growth media and optical indicators, which are in contact with both sides of the porous barrier.
The upper chamber of the container has an opening at the top to allow the introduction of a test sample into the container via its open top. The lower chamber serves as the detection zone in which the optical readings are monitored. The lower chamber's internal volume of liquid is completely confined between the container inside wall and the barrier.
Constructing such a test container presents substantial difficulties. The barrier layer must be fixedly attached about its perimeter to the interior of the container side walls at an intermediate level to define the upper and lower chambers. This can be accomplished by ultrasonic welding, heat welding or any other sealing-attachment process. The use of heat as an attachment method precludes the prior introduction of liquid into the lower or second chamber and other sealing-attachment methods would also be difficult to carry out if liquid was present in the second chamber. The liquid must completely fill the lower chamber, as the presence of any air would interfere with the diffusion process.
In order to completely fill the lower chamber with liquid, a hole can be made in the bottom of the container to allow the introduction of test liquid either through the porous barrier from the top or from the bottom through the hole itself. Achieving complete filling of the lower chamber is difficult. If any residual air is present only part of the liquid in the lower chamber can be in direct contact with the barrier layer, thereby reducing the efficiency of the diffusion of molecules between the two chambers. The hole must thereafter be reliably sealed, leading to manufacturing difficulties when produced in quantity due to the inevitable incidence of leaks in some of the devices.
An object of the present invention is to provide a method that substantially completely evacuates the air in the lower chamber and allows test liquid to completely fill the entire volume of the lower chamber in order to promote maximal diffusion between the lower chamber and upper chamber of a two chamber test container when a test sample is introduced to the upper chamber, so that monitoring can then be effectively carried out by optical monitoring of the lower chamber.